RESEARCH ARTICLE Open Access

Characterization of the mechanism ofaction of lanraplenib, a novel spleentyrosine kinase inhibitor, in models of lupusnephritisChristopher W. Pohlmeyer1* , Ching Shang1, Pei Han1, Zhi-Hua Cui1, Randall M. Jones1, Astrid S. Clarke1,Bernard P. Murray2, David A. Lopez1, David W. Newstrom3, M. David Inzunza3, Franziska G. Matzkies4,Kevin S. Currie5 and Julie A. Di Paolo1

Abstract

Background: B cells are critical mediators of systemic lupus erythematosus (SLE) and lupus nephritis (LN), andantinuclear antibodies can be found in the serum of approximately 98% of patients with SLE. Spleen tyrosine kinase(SYK) is a nonreceptor tyrosine kinase that mediates signaling from immunoreceptors, including the B cell receptor.Active, phosphorylated SYK has been observed in tissues from patients with SLE or cutaneous lupus erythematosus,and its inhibition is hypothesized to ameliorate disease pathogenesis. We sought to evaluate the efficacy andcharacterize the mechanism of action of lanraplenib, a selective oral SYK inhibitor, in the New Zealand black/white(NZB/W) murine model of SLE and LN.

Methods: Lanraplenib was evaluated for inhibition of primary human B cell functions in vitro. Furthermore, theeffect of SYK inhibition on ameliorating LN-like disease in vivo was determined by treating NZB/W mice withlanraplenib, cyclophosphamide, or a vehicle control. Glomerulopathy and immunoglobulin G (IgG) deposition werequantified in kidneys. The concentration of proinflammatory cytokines was measured in serum. Splenocytes wereanalyzed by flow cytometry for B cell maturation and T cell memory maturation, and the presence of T follicularhelper and dendritic cells.

Results: In human B cells in vitro, lanraplenib inhibited B cell activating factor-mediated survival as well as activation,maturation, and immunoglobulin M production. Treatment of NZB/W mice with lanraplenib improved overall survival,prevented the development of proteinuria, and reduced blood urea nitrogen concentrations. Kidney morphology wassignificantly preserved by treatment with lanraplenib as measured by glomerular diameter, protein cast severity, interstitialinflammation, vasculitis, and frequency of glomerular crescents; treatment with lanraplenib reduced glomerular IgGdeposition. Mice treated with lanraplenib had reduced concentrations of serum proinflammatory cytokines. Lanraplenibblocked disease-driven B cell maturation and T cell memory maturation in the spleen.

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© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

* Correspondence: chris.pohlmeyer@gilead.com1Department of Biology, Gilead Sciences, Inc., 333 Lakeside Dr, Foster City,CA 94404, USAFull list of author information is available at the end of the article

BMC RheumatologyPohlmeyer et al. BMC Rheumatology (2021) 5:15 https://doi.org/10.1186/s41927-021-00178-3

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Conclusions: Lanraplenib blocked the progression of LN-like disease in NZB/W mice. Human in vitro and murine in vivodata suggest that lanraplenib may be efficacious in preventing disease progression in patients with LN at least in part byinhibiting B cell maturation. These data provide additional rationale for the use of lanraplenib in the treatment of SLE and LN.

Keywords: Spleen tyrosine kinase, SYK inhibition, B-cell receptor, B-cell signaling, Immunohistochemistry, Systemic lupuserythematosus, Lupus nephritis, Inflammation

BackgroundSystemic lupus erythematosus (SLE) is a chronic hetero-geneous autoimmune disease, driven by an autoimmuneattack on any of several organs, most commonly the skinor kidney. The pathogenesis of SLE is complex, andmany pathways involving various immune cell types aredysregulated. Current pharmacotherapy is tailored to-ward reducing disease activity and preventing end-organmanifestations through broadly immunosuppressivemechanisms. Commonly used treatments include corti-costeroids, azathioprine, cyclophosphamide, and myco-phenolate, though these treatments may have significantside effects that can limit their use. A more targeted in-hibitory profile may reduce off-target effects and im-prove treatment tolerance.Evidence from several studies supports a central role of

B cells in disease pathology operating through several pro-posed mechanisms, including cytokine production, anti-gen presentation to autoreactive T cells, and autoantibodygeneration and production [1–3]. Considerable effort hasbeen spent developing therapeutics to treat SLE, many ofwhich are designed to modulate B cell prevalence or activ-ity [3–5]. Currently, the only targeted therapy approvedfor the treatment of SLE is belimumab, a human mono-clonal antibody with specificity for B-cell activating factor(BAFF); this therapeutic is not approved for severe mani-festations such as lupus nephritis (LN), which occurs in40–80% of patients with SLE [6], and has a poor overallresponse rate [7]. The need for improved treatment ofSLE and LN has driven interest to explore new targets fortherapeutic development. In LN, pathogenesis is driven bydeposition of immune complexes (ICs) in glomeruli; thecomplexes are poorly cleared, leading to acute tissue dam-age and recruitment of immune cells, which propagateadditional damage [8].Spleen tyrosine kinase (SYK) is an integral component

of signaling through multiple immunoreceptor tyrosine-based activation motif (ITAM)-containing immunorecep-tors across several different immune cell types, includingthe B cell receptor (BCR), Fc receptors (FcR), and β2 in-tegrin. Inhibition or deletion of SYK has significant detri-mental immune effects [9]. Through BCR signaling, SYKhas been demonstrated to play a role in the development[10–12], activation [11, 13, 14], and maturation of B cells[15], as well as antibody production and class switching

[15]. SYK has also been demonstrated to be involved inBAFF-mediated B cell survival [16], though its specific rolehas not been resolved [17]. Additionally, SYK has beenshown to be involved in both toll-like receptor (TLR)7-and TLR9-signaling pathways, which can be triggeredupon uptake of ICs [18, 19]. Given the role of SYK in bothBCR and TLR signaling, it has been proposed that inhib-ition of SYK could prevent the maturation of autoreactiveB cells [20].SYK dysregulation has been observed in SLE patients. Pa-

tients with LN have an infiltration of SYK-expressing cells inthe glomeruli [21], and peripheral B cells of SLE patients withactive disease exhibit increased levels of SYK phosphorylation[22]. Upon BCR cross-linking, Chang et al. demonstrated anincrease in SYK phosphorylation in B cells of SLE patientscompared to healthy controls [23]. Additionally, SYK tran-script levels are significantly elevated in keratinocytes of pa-tients with cutaneous lupus erythematosus (CLE) comparedto healthy control subjects [24].Several intracellular SYK inhibitors have been devel-

oped and evaluated in preclinical lupus models, butnone have been evaluated in human clinical trials forSLE or LN [25]. Fostamatinib (R788) successfully ame-liorated lupus-like disease in the New Zealand black/white (NZB/W) [26] and Murphy Roths Large/lympho-proliferation (MRL/lpr) [27] preclinical mouse modelsof SLE and LN, although the inhibitor is nonselectiveand it is unclear if the activity can be solely attributedto inhibition of SYK activity [28]. Fostamatinib andMK-8457 were tested clinically in autoimmune indica-tions and displayed dose-limiting toxicities that limittheir development [29–31]; fostamatinib has been ap-proved for immune thrombocytopenia, though it re-quires monitoring and dose adjustments due totoxicities [32]. Additionally, GSK2646264 is currentlybeing evaluated for topical use in the treatment of CLE(NCT02927457).Lanraplenib is a novel, selective, oral, once-daily intra-

cellular SYK inhibitor [33]. In the current study, we ex-amined the effects of lanraplenib on primary human Bcell functionality and LN-like disease progression in theNZB/W murine model. We characterized the potentialmechanism of action of lanraplenib by analyzing renalfunction, renal histology, systemic cytokine levels, andsplenic immune cell populations.

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MethodsHuman B cell in vitro assaysFrozen human peripheral blood mononuclear cells(PBMCs) from healthy donors or individuals with SLE(AllCells) were treated with lanraplenib or dimethyl sulf-oxide (DMSO) before stimulation. To measure B cellsurvival, B cells were treated with human BAFF (R&DSystems, Inc., Minneapolis MN) or RP10; viability wasmeasured using RealTime-Glo™ reagent (Promega Cor-poration, Madison WI). To measure B cell activation, Bcells were stimulated with 20 μg/mL antihuman F (ab’)2immunoglobulin M (IgM) (SouthernBiotech, Birming-ham, AL) for 16 h; activation was measured by expres-sion of CD69 and CD86 (BD Biosciences, San Jose, CA)measured by fluorescence-activated cell sorting (FACS)(FACSCanto™ II, BD Biosciences).Naïve B cells were isolated from freshly collected

PBMCs (AllCells) by negative selection (STEMCELLTechnologies). They were then treated with lanraplenibor DMSO before stimulation with a stimulation cocktailof F (ab’)2 antihuman immunoglobulin D (IgD) (South-ernBiotech), IL-2 and IL-10 (BioLegend®, San Diego,CA), and human MEGACD40L (Enzo Life Sciences,Inc., Farmingdale, NY) for 7 days. Cells were stained forexpression of CD3, CD19, and CD27 (BioLegend) beforeanalysis on an LSRFortessa™ X-20 (BD Biosciences).Supernatant antibody concentration was measured usinga human antibody isotyping 7-plex immunoassay kit(Invitrogen ProcartaPlex, Carlsbad, CA) read on a FLEX-MAP 3D® (Luminex Corporation, Austin, TX).

NZB/W study: ethical statementThis basic study design and animal usage was approvedby Bolder BioPATH’s Institutional Animal Care and UseCommittee (IACUC) for compliance with regulationsprior to study initiation (IACUC Protocol #BBP-005).

NZB/W study: study designMice were housed at Bolder BioPATH until enrollment inthe study at 28weeks. Mice were assigned to one of 4 groupsof 15 mice each (1 positive, 1 negative control group, 2 ex-perimental groups) by distributing mice by body weight andproteinuria equivalently across groups. All mice were bled byretro-orbital sinus, at ages 28, 34, and 40weeks for plasmafor anti–dsDNA antibody analysis (Alpha Diagnostic) and at29, 31, and 35weeks for PK plasma. Urine was collectedfrom all mice weekly to measure proteinuria by dipstickmeasurement. Body weight was measured weekly. Food con-sumption was measured daily. An overview of the study de-sign is shown in Fig. 2.

NZB/W study: experimental proceduresAnimals were housed 3–5/cage in filtered, pie-shapedpolycarbonate cages (Animal Care Systems, Inc.) with

wood chip bedding and suspended food and water bot-tles. Animal care including room, cage, and equipmentsanitation conformed to the guidelines cited in the Guidefor the Care and Use of Laboratory Animals (Guide, 2011)and the applicable standard operating procedures ofBolder BioPATH. During the acclimation and study pe-riods, animals were housed in a laboratory environmentwith temperatures ranging 67–76 °F and relative humidityof 30–70%. Automatic timers provided 12 h of light and12 h of dark. Animals were allowed access ad libitum toHarlan Teklad Rodent Chow and fresh municipal tapwater. Mice were dosed orally with lanraplenib (0.075% or0.25%) or vehicle-administered ad libitum in chow. Posi-tive controls were treated with cyclophosphamide (5mg/kg) administered daily intraperitoneally.

NZB/W study: experimental animalsFemale NZBWF1/J mice were purchased from The Jack-son Laboratory (Bar Harbor, ME) at 20 weeks of age;additional female mice (n = 15) that were 14 weeks oldon arrival at study week 37 were obtained and used aspredisease FACS analysis controls. Mice weighed from30 to 55 g from arrival at Bolder BioPATH until studyinitiation. At the end of study, mice were anesthetizedwith Isoflurane (VetOne) and bled by cardiac puncturefor terminal plasma, serum, and whole blood. Spleensand kidneys were also harvested and weighed immedi-ately. Spleens were processed and immunophenotypedby flow cytometry. Kidneys were fixed in 10% neutralbuffered formalin before being embedded into paraffin.Kidney sections were stained with hematoxylin and eosinfor histologic analysis. For mice that died or were eutha-nized prior to study termination, terminal clinical scoreswere carried through to study termination for analysispurpose.

Serum cytokine concentrationSerum cytokine concentrations were measured with amurine cytokine and chemokine 36-plex immunoassay(Invitrogen ProcartaPlex). Samples were analyzed usinga FLEXMAP 3D (Luminex Corporation). Measurementsof concentrations of proinflammatory cytokines wereanalyzed.

Splenocyte preparation and staining for flow cytometryCells were stained for FcR blocking antibodies (BioLe-gend) before staining with Bcl6, CD3, CD4, CD8,CD11b, CD11c, CD19, CD23, CD44, CD45R/B220,CD62L, CD93, CD138, CD172a, CXCR5, IgM, MHCII(BioLegend), and IgD (Invitrogen) in 4 separate panels.Stained samples were analyzed on a FACSCelesta, anLSR II, or an LSRFortessa X-20 (BD Biosciences). Datawere analyzed using FlowJo software.

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RNA extraction and gene expression analysisTotal RNA was extracted from formalin fixed paraffin-embedded scrolls of mouse kidney (Acepix Bioscience,Inc., Hayward, CA). The expression of 93 target genesand 3 housekeeping genes (RPL19, RPL0, and ACTB)were analyzed with standard Taqman assays using Flui-digm Biomark RT-qPCR (bioanalytic and single-cell coreat UT Health at San Antonio). Cycle threshold (Ct) datawere collected by GE 96 × 96 Standard v2 software. Ctvalues for each transcript were normalized using thegeometric mean of ACTB, RPL19, and RPLP0 Ct values.P values were calculated by Mann-Whitney U test.Nominal P values are presented.

Kidney H&E histopathology measurementsTwenty glomeruli per sample were measured to determinemean glomerular diameter and crescent score. Scoringwas performed according to methodology previouslyestablished [34]. Specifically: Glomerular diameter wasscored as follows: 0: > 65 μm; 0.5: 60–70 μm; 1: 71–80 μm;2: 81–90 μm; 3: 91–100 μm; 4: 101–110 μm; 5: > 110 μm.Crescent percentage was scored as follows: 0: no crescents;0.5: single glomerulus with crescent; 1: 2–4% of glomeruliwith crescents; 2: 5–10% of glomeruli with crescents; 3:11–25% of glomeruli with crescents; 4: 25–50% of glom-eruli with crescents; 5: > 50% of glomeruli with crescents.Protein cast severity was scored as followed: 0: normal tu-bules; 0.5: affects 1–2% of cortex; 1: affects 3–10% of cor-tex; 2: affects 11–25% of cortex; 3: affects 26–50% ofcortex; 4: affects 51–75% of cortex; 5: affects > 75% of cor-tex. Interstitial inflammation was scored as follows: 0: noinflammation; 0.5: small focal area of mononuclear cells inpelvis only; 1: occasional small focal accumulation ofmononuclear cells affecting > 10% of total interstitium; 2:multifocal mononuclear infiltrates affecting 10–25% ofcortex area; 3: multifocal mononuclear infiltrates affecting26–50% of cortex area; 4: multifocal mononuclear infil-trates affecting 51–75% of cortex area; 5: diffuse mono-nuclear infiltration affecting 75–100% of cortex area.Vasclitis was scored as follows: 0: no vasculitis; 0.5: onevessel with minimal perivascular infiltrate; 1: 1–2 foci ofperivascular infiltrates; 2: 3–4 foci of perivascular infiltratewithout necrosis; 3: 5–6 foci of perivascular infiltrate withor without necrosis of vessel wall; 4: 7–8 foci of perivascu-lar infiltrate with or without necrosis of vessel wall; 5: > 8foci of perivascular infiltrate with extensive necrosis.Summed scores represent sum of each of 5 individuallymeasured criteria above.

IgG staining and scoringKidney sections were stained with Alexa Fluor 647 con-jugated anti-mouse IgG antibody (Cell Signaling Tech-nology®, 4410S) and DAPI. IgG deposition in glomeruliwas scored into 4 grades based on the amount of

fluorescence signal observed in each glomerulus: Grade1: < 25% deposit; Grade 2: 25–49% deposit; Grade 3: 50–74% deposit; Grade 4: ≥75% deposit. Approximately 200glomeruli were scored from each mouse.

Statistical analysisStatistics were calculated in Prism (GraphPad SoftwareInc.) unless otherwise specified. Pairwise comparisonswere made by testing for normal distribution of data.Data normality was tested by D’Agnostine-Pearsonomnibus normality test. Normally distributed data wereanalyzed by ANOVA and non-normally distributed datawere analyzed by a Kruskal-Wallis test. ANOVA wascorrected for multiple comparisons by the Holm-Sidakmethod; Kruskal-Wallis was corrected with a Dunn’smultiple comparison test. For analyses with samplesbelow the limit of detection, the limit of detection wasused as the quantified value. Effective concentration(EC)50 values were calculated using a 4-parameter vari-able slope nonlinear regression fitted by least squares.Nonlinear best fit regressions were calculated usingGraphPad Prism.

ResultsSYK inhibition blocks B cell functions in vitroThe effect of SYK inhibition on primary human B cellswas characterized in vitro. In culture, human B cells dierapidly; however, when stimulated with recombinantBAFF, their survival is prolonged (Fig. 1a). Lanraplenibtreatment of B cells prior to BAFF stimulation dose-dependently reduced B cell survival with an EC50 value of130 nM (Fig. 1a and b). Interestingly, lanraplenib inhibitedBAFF-mediated B cell survival to a level below the un-stimulated control (Fig. 1a), demonstrating that lanraple-nib can inhibit basal survival signaling in human B cells[35]. Lanraplenib also inhibited BAFF-mediated survival ofmouse BALB/c splenic B cells in a dose-dependent fashion(see Supplemental Fig. 1A, Additional File).As SYK is a critical component of the BCR signaling

cascade, the effect of lanraplenib on BCR-mediated Bcell activation was measured. Primary human B cells iso-lated from the blood of healthy donors or individualswith SLE were treated with lanraplenib, and the expres-sion of CD69 was measured following BCR engagement.We observed a dose-dependent inhibition of CD69 ex-pression with EC50 values of 298 nM and 340 nM, re-spectively (Fig. 1c).To test the effect of lanraplenib on B cell maturation

in vitro, lanraplenib-treated CD27-naïve human B cellswere stimulated with a cocktail of interleukin (IL) 2, IL10,and CD40L, and a BCR-stimulating polyclonal antibody (F(ab’)2 anti-IgD). B cell maturation was blocked by SYK in-hibition as measured by expression of CD27 with an EC50

value of 245 nM (Fig. 1d). Additionally, lanraplenib

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inhibited the secretion of IgM in a dose-dependent fashionwith an EC50 value of 216 nM (Fig. 1e). Similar resultswere observed when murine splenic B cells were stimu-lated with a cocktail of IL4, CD40L, and BCR-stimulatingmonoclonal antibody (see Supplemental Figs. 1B and C,Additional File).

Lanraplenib ameliorates LN-like disease in NZB/W miceTo evaluate the efficacy of lanraplenib in an in vivo sys-tem, lanraplenib was evaluated in the NZB/W murinepreclinical model of SLE. These mice recapitulated sev-eral clinical elements of SLE [36]. The study scheme forevaluating lanraplenib in NZB/W mice is depicted inFig. 2a. Mice dosed at 0.075 and 0.25% in chow had cal-culated average SYK target coverage (AUC) of approxi-mately 50 and 80%, respectively as measured by apervanadate PD assay; due to the nature of continuousfood consumption and subsequent continuous dosing,target coverage remained consistent over the 24-hperiod measured (data not shown). The lanraplenib(0.075%)-treated group did not show a statistically sig-nificant difference in survival compared to the vehicle

control group at week 40 (Fig. 2b). Groups treated witheither lanraplenib (0.25%) or cyclophosphamide (5 mg/kg/day) showed a statistically significant improvement inoverall survival at week 40 compared to the vehicle-treated group (P = 0.044 and 0.010, respectively).NZB/W mice develop significant glomerulonephritis

and proteinuria. While approximately 90% of mice in thevehicle-treated group developed proteinuria (≥100mg/dL)by week 40, lanraplenib (0.25%) treatment prevented thedevelopment of proteinuria to a similar extent as cyclo-phosphamide (5mg/kg/day) treatment (Fig. 2c). Due tothe inherent limitations associated with measuring pro-teinuria by dipstick measurements, we also measuredblood urea nitrogen to characterize kidney activity. Bothlanraplenib (0.25%) and cyclophosphamide treatment (5mg/kg/day) significantly reduced blood urea nitrogenlevels, another indicator of improved kidney function (Fig.2d). Treatment with lanraplenib (0.25%) or cyclophospha-mide reduced kidney weight, spleen weight, and bloodcholesterol levels, suggesting the general improvement oflupus disease phenotypes (see Supplemental Figs. 2A, B,and C, Additional File). While treatment with either

Fig. 1 Lanraplenib inhibits human B-cell survival, activation, maturation, and antibody production of in vitro. a-b Lanraplenib-treated B cells fromhealthy donors (HD) were stimulated with recombinant B-cell activating factor (BAFF) (250 ng/mL) for 48 h. Survival was measured by RealTimeGlo. Comparison of unstimulated and BAFF-stimulated B cells with and without lanraplenib treatment are shown in a. Survival of untreated cellswithout BAFF stimulation was subtracted from all wells to generate dose response curve in b. EC50: 130 nM; n = 6. c Lanraplenib-treated B cellsisolated from blood of HD or individuals with SLE were stimulated with F (ab’)2 anti-IgM antibody (20 μg/mL) for 16 h. Activation was measuredby the expression of CD69. EC50: 298 nM (HD), 340 nM (SLE); n = 3 donors per group. d Lanraplenib-treated naïve B cells (CD27−) from HD werestimulated with a cocktail of interleukin (IL)-2 (50 U/mL), IL-10 (10 ng/mL), CD40L (100 ng/mL), and F (ab’)2 anti-IgD antibody (1 μg/mL) for 7 days.Maturation was measured by the expression of CD27. EC50: 245 nM; n = 4. e Antibody production was measured by the concentration of IgM insupernatant samples from c. EC50: 216 nM; n = 4. Error bars represent standard error of the mean (SEM). * Indicates P < 0.05 as calculated byrepeated measures one-way ANOVA

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lanraplenib (0.25%) or cyclophosphamide improved over-all survival and kidney function, mice treated with cyclo-phosphamide lost more body weight compared to thevehicle-treated mice, whereas mice treated with lanraple-nib (0.25%) lost less weight than vehicle-treated mice (Fig.2e), suggesting that lanraplenib was better tolerated bythese mice.

Kidney morphology is improved by SYK inhibitionRenal histology was evaluated to better characterize the ef-fect of lanraplenib treatment. Because NZB/W mice spon-taneously develop LN-like disease, young, prediseasedmice were used as controls. Glomerular diameter, proteincast severity, interstitial inflammation, vasculitis, and fre-quency of glomerular crescents were measured in sectionsof renal tissue harvested from mice from each treatmentgroup (see Supplemental Fig. 3). Vehicle-treated mice hadsignificantly increased glomerular diameter compared topredisease mice (Fig. 3a). Treatment with lanraplenib(0.075%) had minimal effect on glomerular diameter.Treatment with lanraplenib (0.25%) or cyclophosphamidesignificantly reduced glomerular diameter. The summedscores for the kidney morphologic features measured,shown in Fig. 3b, are significantly increased in vehicle-treated mice as compared to the predisease mice. The lan-raplenib (0.075%) treatment group showed no improve-ment in kidney score, whereas the kidney scores weresignificantly reduced in both the lanraplenib (0.25%) and

cyclophosphamide treatment groups toward prediseaselevels.As antibody deposition in the kidney can drive neph-

ritis and glomerular destruction, IgG deposition in glom-eruli across treatment groups was quantified by gradingthe degree of IgG deposition into 4 categories (grade 1–4) (see Supplemental Fig. 3, Additional File). Representa-tive glomerular IgG staining in each treatment is shownin Fig. 3c. We observed an increase in IgG deposition inglomeruli based on the presence of disease (vehicle-treated group vs predisease group) (Fig. 3d). Lanraplenib(0.075%) treatment did not reduce glomerular IgG de-position, whereas lanraplenib (0.25%) or cyclophospha-mide treatment significantly reduced glomerular IgGdeposition as measured by IgG grade score (Fig. 3d).

Lanraplenib normalizes transcription of genes in kidneysectionsTo further characterize the effect of lanraplenib on thekidney, renal transcripts of select genes that encode cy-tokines and proteins involved in apoptosis, apoptotic cellclearance, and structural integrity were quantified. Sev-eral of the transcripts were significantly more prevalentin renal tissue harvested from vehicle-treated mice com-pared to those of predisease mice (see SupplementalFig. 4A, Additional File) and were significantly lessprevalent in the renal tissue harvested from either thelanraplenib (0.25%)- or cyclophosphamide-treated mice

Fig. 2 Lupus-like phenotype in NZB/W mice is ameliorated by lanraplenib treatment. a Experimental study design to test the efficacy of lanraplenib.Lanraplenib was formulated in chow at the indicated percentages. Proteinuria was measured every week starting at week 28. b Kaplan-Meier curveshowing the percentage of surviving mice at each time point; n = 15 mice per group at week 28. c Percentage of mice with proteinuria (≥ 100mg/dL)at each time point. Treatment groups are stylized as in b. d Blood urea nitrogen measurement at week 40. Each point represents an individual mouseat the time of sacrifice. Mean ± SEM are indicated for each treatment group. Statistics were calculated comparing the vehicle control group to eachother group using a Kruskal-Wallis test. e Mean body weight of mice in each group ± SEM. Treatment groups are stylized as in b. Food consumptionwas measured daily; no differences in food consumption were observed across any treatment groups. * Indicates P < 0.05. *** indicates P < 0.0005.†Indicates blood draws for dsDNA Ab measurement. ‡Indicates blood draws for PK measurement

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compared to those of the vehicle-treated mice. A similarmagnitude of change in the prevalence of transcriptswas observed when the vehicle-treated group was com-pared to each of the predisease, lanraplenib (0.25%)-treated, and cyclophosphamide-treated groups (see Sup-plemental Fig. 4B, Additional File).

SYK inhibition reduces serum proinflammatory cytokineconcentrationsTo gain an understanding of the overall phenotype of dis-ease amelioration with lanraplenib treatment, the concen-tration of 18 proinflammatory cytokines in the serum ofNZB/W mice was measured at age 40 weeks. Of the 18 cy-tokines measured, the concentrations of 7 were below thelimit of detection (see Supplemental Fig. 5, AdditionalFile). Treatment with lanraplenib (0.075%) had no effecton serum concentrations of monocyte chemotactic pro-tein (MCP)-1 or tumor necrosis factor (TNF)-α (Fig. 4a);however, the concentration of both cytokines was signifi-cantly lower in the lanraplenib (0.25%) and cyclophospha-mide treatment groups. Treatment with either lanraplenib(0.25%) or cyclophosphamide showed an overall trend to-ward a reduction in the proinflammatory cytokine

concentrations. A statistically significant reduction wasobserved in 5 serum cytokines with lanraplenib (0.25%)and 9 with cyclophosphamide, suggesting that lanraplenibhas a more targeted inhibitory profile while cyclophospha-mide potentially has a broader immunosuppressive effect.A heat map comparing the fold-change of the geometricmean of cytokine concentration between the vehicle-treated group to each of the lanraplenib (0.075%)-, lanra-plenib (0.25%)-, and cyclophosphamide-treated groups isshown in Fig. 4b.

Lanraplenib inhibits maturation of splenic B cells in NZB/W miceTo determine if SYK inhibition could inhibit B cell mat-uration in vivo, the presence of B cells in different stagesof maturation in the spleens of NZB/W mice was quan-tified. Figure 5a shows a simplified schematic of thestages of B cell maturation that were measured and thecell surface markers used to identify each population byflow cytometry. Figure 5b shows the gating scheme usedto identify B cells in the different stages of maturation.There was no detectable change in the abundance of im-mature B cells due to disease progression; however,

Fig. 3 Kidney morphology is improved by lanraplenib treatment in NZB/W mice. a Average glomerulus diameter of kidney sections. Each pointrepresents an individual mouse at the time of sacrifice. b Summed histology score of kidney sections. c Representative glomerular IgG stainingfor each group. Green: anti-IgG staining; red: DAPI. Scale bar represents 100 μm. d Summary of glomerular IgG staining. Scoring criteria arespecified in Fig. S3. Mean ± SD are indicated for each treatment group. Statistics were calculated comparing the vehicle control group to eachother group using either an ANOVA or Kruskal-Wallis test. *Indicates P < 0.05. ** Indicates P < 0.005. *** Indicates P < 0.0005

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vehicle-treated mice had a significantly increased countof plasma cells compared to predisease mice (Fig. 5c).Treatment with lanraplenib (0.25%) or cyclophospha-mide did not have an effect on immature B-cell countbut significantly reduced the development of plasmacells. Supplemental Figs. 6A and B, Additional File, showthe effect of disease and treatment with lanraplenib(0.25%) or cyclophosphamide within each stage of B cellmaturation shown in Fig. 5a. Overall, lanraplenib treat-ment modulated the abundance of B cells in later stagesof maturation without affecting the prevalence of B cellsin earlier stages of maturation.Additionally, T follicular helper (Tfh) cells were found

to be more abundant in vehicle-treated mice comparedto predisease mice (Fig. 5c). Treatment with lanraplenib(0.25%) or cyclophosphamide reduced the number ofTfh cells in the spleen. The disease was also observed toaffect the ratio of naïve to memory CD4 and CD8 Tcells, as well as the prevalence of CD11b+ dendritic cellswithin the spleen; treatment with lanraplenib (0.25%) orcyclophosphamide had restorative effects on all of these

populations (see Supplemental Fig. 7, Additional File)back to predisease levels.

DiscussionIn this study, we sought to evaluate the efficacy andcharacterize the mechanism of action of lanraplenib, aselective oral SYK inhibitor, in human B cells andin vivo in the NZB/W murine model of SLE. Our resultsdemonstrate that lanraplenib-mediated inhibition ofSYK can block in vitro B cell activation and maturationof isolated primary human B cells, which are believed tobe a driver of disease. Additionally, lanraplenib blockedthe progression of LN-like disease in NZB/W mice.Altogether, these data suggest that the effect of lanraple-nib in preventing disease progression was at least in partdue to inhibition of B cell maturation.B cells are critical mediators of SLE pathology and

have generated research interest. B cells have multiplefunctions that affect SLE pathogenesis, including cyto-kine production, antigen presentation to autoreactive Tcells, and autoantibody generation and production [1–3].

Fig. 4 Serum proinflammatory cytokine concentrations are reduced in lanraplenib-treated NZB/W mice. a Geometric mean (± geo standarddeviation) serum concentration of MCP-1 (left) and TNF-α (right) of individual mice. Each point represents an individual mouse. Limit of detectionof assay is represented by a dashed line. Open circles indicate serum concentrations below the limit of detection. Statistics were calculatedcomparing the vehicle control group to each other group using Kruskal-Wallis tests. b Heat map summarizing the geometric mean fold-changecomparing the vehicle control group to each indicated treatment group. *Indicates P < 0.05. **Indicates P < 0.005. ***Indicates P < 0.0005

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Antinuclear antibody (ANA) production is a hallmark ofSLE; the most commonly found ANA recognizes dsDNAand can be found in the serum of 30–70% of patientswith SLE [4, 37, 38]. ANAs can form ICs with nuclearmaterials released during apoptosis [39, 40], necrosis[39], and NETosis [41]. In healthy individuals, ICs arebound by complement proteins and are quickly cleared[42]; however, many SLE patients have defects in one ormore complement pathway components [43], leading toreduced clearance of ICs and resultant type III hypersen-sitivity [44].In the present study, we did not observe a change in

serum dsDNA antibody concentrations between 28 and40 weeks of age with disease progression. It is likely that

the effects of lanraplenib or cyclophosphamide on serumdsDNA antibody concentration were negligible due tothe initiation of treatment after dsDNA antibody pro-duction had already plateaued. Bahjat et al. observed asimilar lack of dsDNA antibody change in vehicle-treated NZB/W after 28 weeks of age [26]. We have pre-viously tested the effects of a molecule in the same classas lanraplenib in the MRL/lpr murine model of lupus.Between 14 and 20 weeks of age, vehicle-treated MRL/lpr mice showed significantly increased serum dsDNAantibody levels; however, mice treated with either a SYKinhibitor or cyclophosphamide had significantly lowerserum dsDNA antibody concentration compared to thevehicle-treated mice at week 20 (see Supplemental Fig. 8,

Fig. 5 B cell maturation is prevented by lanraplenib in the spleens of NZB/W mice. a Schematic of B cell maturation in the spleen. Surface markers used in thisstudy to define each phase of B cell maturation are shown. b Representative flow cytometry gating scheme. c Geometric mean (± geo standard deviation) ofthe abundance of immature B cells (left), plasma cells (middle), and Tfh cells (right) within the spleens of mice at the end of the study. Cell counts werenormalized to spleen mass. Each point represents an individual mouse. Statistics were calculated comparing the vehicle control group to each other groupusing either an ANOVA or Kruskal-Wallis test. *Indicates P <0.05. ***Indicates P <0.0005

Pohlmeyer et al. BMC Rheumatology (2021) 5:15 Page 9 of 13

Additional File). These data suggest that SYK inhibitioncan prevent the development of autoantibodies in vivo.Additionally, by reducing B cell activation and matur-ation, inhibition of SYK may prevent the intra- andinterantigenic epitope spreading observed in SLE pa-tients [45–48].BCR signaling is necessary for B cell activation and

maturation into antibody-producing cells [49], and SYKis a central player in BCR signal transduction. Uponbinding to doubly phosphorylated ITAM of the BCR [50,51], SYK is auto- [52] and transphosphorylated [53, 54],and recruits signaling molecules including BLNK [55,56], PI3K [57, 58], PLC-γ [59, 60], and VAV [61, 62]. In-hibition of SYK phosphorylation results in dissociationof SYK from the ITAM and internalization of the BCR[63]. Conditional SYK knockout mice have defects in Bcell development [10, 12] and produce decreased levelsof antigen-specific antibodies after exposure to antigen[15]. In this study, we observed an inhibition in matur-ation of B cells within the spleens of NZB/W micetreated with lanraplenib (0.25%).SYK has a role in many biological pathways that could

contribute to SLE pathogenesis, and its modulation mayhave a therapeutic benefit beyond affecting B cell functionand maturation. Through its dual Src homology 2 do-mains, SYK has been demonstrated to interact withITAMs downstream of integrins [64, 65], FcR [66], andother signaling molecules [67–69]. SYK is expressed inmany different cell types that have ITAM-containing sig-naling receptors, including neutrophils, conventional den-dritic cells (DCs), plasmacytoid DCs (pDCs), and others.In neutrophils, SYK signaling occurs after β2 integrin

activation during rolling adhesion [70, 71] and transcel-lular migration [72]. In addition, proinflammatory cyto-kines and chemokines, including MCP1, MCP2, andmacrophage inflammatory protein (MIP)-1α, are in-volved in neutrophil recruitment; B cells and myeloidcells can produce MCP1, MCP2, and MIP-1α via TLR7and TLR9 signaling pathways after endocytosis orphagocytosis of nucleic acid-containing ICs through FcRmolecules [73–78]. Furthermore, stimulation of pDCswith serum isolated from SLE patients can induce pro-duction of several proinflammatory cytokines, includingTNF-α, through the uptake of nucleic acid-containingICs via FcR molecules [79, 80]. SYK inhibition has beendemonstrated to block FcR-dependent IC uptake byclassical DCs [81]. In the present study, treatment withlanraplenib reduced the serum concentration of MCP1,MIP-1α, and TNF-α in NZB/W mice (Fig. 4b).The T cell receptor (TCR) signals through several

ITAMs [82, 83], and it has been proposed that SYK playsa role in TCR signaling in SLE patients [84–87]. How-ever, the role of SYK in TCR signaling in NZB/W miceis unknown. The observed effects on splenic T cells with

lanraplenib treatment (decreased T cell memory matur-ation and reduced Tfh abundance) are likely secondaryeffects and not attributable to direct SYK inhibition of Tlymphocyte signaling. We hypothesize that the inhibitionof proinflammatory cytokine production observed in thisstudy reduced CD11b+ DC activation and subsequentactivation of T cells and differentiation into Tfh [88, 89].This could further support the role of SYK inhibition intreatment of Sjögren’s syndrome.SYK has a central role in diverse functional activities

across multiple cell types, including BCR signal trans-duction, neutrophil adhesion, and FcR-mediated endo-cytosis. In this study, we observed a reduction in splenicB cell maturation with lanraplenib treatment.

ConclusionThese results demonstrate that lanraplenib has a pro-tective role against the development of LN-like diseasein NZB/W mice. SYK inhibition may have multiple pro-tective roles in SLE and LN, including but not limited toinhibition of B cell maturation. Characterization of Bcells in various stages of maturation may be beneficial tomonitor in clinical studies of lanraplenib.

Supplementary InformationThe online version contains supplementary material available at https://doi.org/10.1186/s41927-021-00178-3.

Additional file 1: Supplemental Figure1. Lanraplenib inhibits murineB cell survival, maturation, and antibody production in vitro. (A)Lanraplenib-treated splenic CD43− B cells were stimulated with recombin-ant BAFF (1 ng/mL) for 72 h. Survival was measured by RealTime Glo. Sur-vival measurement of untreated cells without BAFF stimulation wassubtracted from all wells. EC50: 121 nM. n = 3. (B) Lanraplenib-treatedsplenic CD43- B cells were stimulated with a cocktail of IL4 (10 U/mL),CD40L (200 ng/mL), and F (ab’)2 anti-IgD antibody (1 μg/mL) for 7 days.Maturation was measured by the expression of CD27. EC50: 169 nM. n =3. (C) Antibody production was measured by the concentration of IgM insupernatant samples from C. EC50: 462 nM. n = 3. Error bars represent SD.Supplemental Figure2. Improved phenotype of NZB/W F1 mice withlanraplenib treatment. Kidney weight (A), spleen weight (B), and bloodcholesterol levels (C) of each group at time of sacrifice. Each point repre-sents an individual mouse. Mean ± SD are indicated for each treatmentgroup. Statistics were calculated comparing the vehicle control group toeach other group using either ANOVA or Kruskal-Wallis tests. *P < 0.05,**P < 0.005, ***P < 0.0005. Supplemental Figure 3. H&E images andglomerular IgG deposition scoring. (A) Representative H&E images foreach treatment group. G: glomeruli; V: vasculitis; large arrow: protein castsin tubules. (B) Glomerular IgG staining was scored by eye read andbinned into 4 scores. Grade 1: < 25% deposit; Grade 2: 25–49% deposit;Grade 3: 50–74% deposit; Grade 4: ≥75% deposit. Green: anti-IgG staining;red: DAPI. Supplemental Figure 4. Transcriptional changes in renalsamples of NZB/W F1 mice are restored to prediseased levels after lanra-plenib treatment. (A) Volcano plot showing fold-change and nominal Pvalues of transcripts of apoptotic proteins, proteins involved in apoptoticcell clearance, cytokines, and structural proteins. Each point represents anindividual gene transcript. Horizontal line represents a 10− 4 P value cutoff.Vertical lines represent a 2-fold change cutoff. (B) Heatmap summarizingfold-change comparing each group to the vehicle control group. Tran-scripts quantified are specified below plot. Supplemental Figure 5.Changes in serum proinflammatory cytokine concentrations inlanraplenib-treated NZB/W F1 mice. Geometric mean (± Geo SD) serum

Pohlmeyer et al. BMC Rheumatology (2021) 5:15 Page 10 of 13

concentration of all cytokines that were above (A) or below the limit ofdetection (B). Each point represents and individual mouse. Statistics werecalculated comparing the vehicle control group to each other groupusing Kruskal-Wallis tests. Supplemental Figure 6. Splenic B cells have aless-matured phenotype in lanraplenib-treated NZB/W F1 mice. Geometricmean (± Geo SD) of abundance of B cells in each step of the maturationprocess within the spleens of mice at the end of study. Cell counts werenormalized to spleen mass. Each point represents an individual mouse.Statistics were calculated comparing the vehicle control group to eachother group using either ANOVA or Kruskal-Wallis tests. *P < 0.05, **P <0.005, ***P < 0.0005. Supplemental Figure 7. Reduced abundance of Tand dendritic cells in the spleens of lanraplenib-treated NZB/W F1 mice.Mean ratio of naïve:memory CD4+ (left) and CD8+ (middle) at the end ofstudy. Geometric mean (± Geo SD) of abundance of CD11b+ DC (right)within the spleens of mice at the end of study. Cell counts were normal-ized to spleen mass. Each point represents an individual mouse. Statisticswere calculated comparing the vehicle control group to each othergroup using an ANOVA test. *P < 0.05, **P < 0.005, ***P < 0.0005. Supple-mental Figure 8. SYK inhibition blocks dsDNA antibody development inMRL/lpr mice. Average dsDNA antibody titers in each treatment group(n = 15 animals per group). Treatment was initiated at age 14 weeks .*P < 0.05, ***P < 0.0005.

AbbreviationsANA: Anti-nuclear antibody; BAFF: B cell activating factor; BCR: B cellreceptor; CLE: Cutaneous lupus erythematosus; Ct: Cycle threshold;DC: Dendritic cell; DMSO: Dimethyl sulfoxide; dsDNA: Double-stranded DNA;EC: Effective concentration; FACS: Fluorescence-activated cell sorting;FcR: Fragment, crystallizable receptor; IC: Immune complexes;IgD: Immunoglobulin D; IgG: Immunoglobulin G; IgM: Immunoglobulin M;IL: Interleukin; ITAM: Immunoreceptor tyrosine-based activation motif;LN: Lupus nephritis; MCP: Monocyte chemotactic protein; MIP: Macrophageinflammatory protein; MRL/lpr: Murphy Roths Large/lymphoproliferation;NZB/W: New Zealand black/white; PBMCs: Peripheral blood mononuclearcells; PD: Pharmacodynamic; pDC: Plasmacytoid dendritic cells; SEM: Standarderror of the mean; SLE: Systemic lupus erythematosus; SYK: Spleen tyrosinekinase; TCR: T cell receptor; Tfh: T follicular helper; TLR: Toll-like receptor;TNF: Tumor necrosis factor

AcknowledgmentsThis work was solely supported by Gilead Sciences, Inc. The authors wish tothank Bolder BioPATH and ClinImmune Labs for data analysis, as well as G.Min-Oo for her scientific input, advice, and critical evaluation of the manu-script. Editorial support was provided by Impact Communication Partners(New York, NY, US).

Authors’ contributionsCP, ZHC, CS, PH, and JDP developed the concept and designed the research;CP, CS, PH, ASC, RJ, BPM, MDI, DL, and DN analyzed the data; CP, CS, andJDP drafted the manuscript; KSC, FM, and JDP edited and revised themanuscript. All authors have read and approved the manuscript.

FundingStudy was funded by Gilead Sciences, Inc.; all authors are employees ofGilead Sciences and were involved in study design and collection, analysis,and interpretation of the data.

Availability of data and materialsThe datasets used and/or analysed during the current study are availablefrom the corresponding author on reasonable request.

Ethics approval and consent to participateHuman sample collection.Prior to blood collection, all individuals gave written informed consent incompliance with the Institutional Review Board-approved protocols for STEMCELL™ Technologies (Vancouver, BC) or AllCells® (Alameda, CA).Animal study.This basic study design and animal usage was approved by Bolder BioPATH’sInstitutional Animal Care and Use Committee (IACUC) for compliance withregulations prior to study initiation (IACUC Protocol #BBP-005).

Consent for publicationNot applicable.

Competing interestsAll authors are employees of Gilead Sciences, Inc. CWP and CS hold stock inand receive research funding from the company. CS, ASC, RMJ, DAL, DWN,FGM, KSC, and JAD hold stock in the company. ZHC, BPM, and MDI declarethey have no competing interests.

Author details1Department of Biology, Gilead Sciences, Inc., 333 Lakeside Dr, Foster City,CA 94404, USA. 2Department of Drug Metabolism, Gilead Sciences, Inc.,Foster City, CA, USA. 3Department of Nonclinical Safety and Pathobiology,Gilead Sciences, Inc., Foster City, CA, USA. 4Department of Clinical Research,Gilead Sciences, Inc., Foster City, CA, USA. 5Department of Chemistry, GileadSciences, Inc., Foster City, CA, USA.

Received: 27 February 2020 Accepted: 22 January 2021

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